Carbonic anhydrase inhibitors: Synthesis and topical intraocular pressure lowering effects of fluorine-containing inhibitors devoid of enhanced reactivity

Article abstract of DOI:10.1021/jm031116j

Polyfluorinated carbonic anhydrase inhibitors (CAIs) show very good inhibitory properties against carbonic anhydrase (CA) and excellent in vivo antiglaucoma properties after topical administration in rabbits. Still, the pentafluorinated compounds reported previously by this group (Scozzafava et al. J. Med. Chem. 2000, 43, 4542-4551) showed high reactivity with thiol groups of cysteine, glutathione, and presumably other proteins containing such moieties, which may lead to severe ocular side effects. Here, we report an approach for obtaining fluorinated CA inhibitors without the undesired enhanced reactivity. Thus, new compounds have been obtained by attaching moieties with reduced reactivity toward aromatic substitution reactions to the molecules of aromatic/heterocyclic sulfonamides possessing derivatizable amino moieties. The employed tails of the 2,3,5,6-tetrafluorobenzoyl, 2,3,5,6-tetrafluophenylsulfonyl, and pentafluorophenylureido types induced excellent CA inhibitory properties in the new reported sulfonamides, mainly against the isozymes involved in aqueous humor secretion, CA II and CA IV, whereas affinity for CA I was lower. Several low-nanomolar CA II inhibitors were detected, which did not react with cysteine or glutathione, in contrast to the corresponding perfluorinated compounds previously reported. These derivatives also showed a potent reduction of the intraocular pressure (IOP) in hypertensive rabbits, amounting to 13-21 mmHg at 1 h postadministration (compared to 5 mmHg obtained with dorzolamide, a clinically used drug), and the decreased IOP was maintained for 4-5 h after the administration. These compounds constitute valuable candidates for obtaining topically acting antiglaucoma CA inhibitors of a new generation, with enhanced efficacy, prolonged duration of action, and reduced side effects.

Full text of DOI:10.1021/jm031116j

2796
J . Med. Chem. 2004, 47, 2796-2804
Ca r bon ic An h yd r a se In h ibitor s: Syn th esis a n d Top ica l In tr a ocu la r P r essu r e
Low er in g Effects of F lu or in e-Con ta in in g In h ibitor s Devoid of En h a n ced
Rea ctivity
Xavier de Leval,†,‡ Monica Ilies,† Angela Casini,† J ean-Michel Dogne´,†,‡ Andrea Scozzafava,† Emanuela Masini,§
Francesco Mincione,# Michele Starnotti,| and Claudiu T. Supuran*,†
Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Universita` degli Studi di Firenze, Via della Lastruccia 3,
50019 Sesto Fiorentino, Florence, Italy, Department of Medicinal Chemistry, Natural and Synthetic Drugs Research Centre,
University of Lie`ge, 1, av. de l’Hoˆpital, B36 Sart-Tilman, B-4000 Lie`ge, Belgium, Department of Preclinical and Clinical
Pharmacology, University of Florence, Viale G. Pieraccini 6, 50139 Florence, Italy, U.O. Oculistica Az. USL 3, Val di Nievole,
Ospedale di Pescia, Pescia, Italy, and Clinica Oculistica, Universita` degli Studi di Firenze, Viale Morgagni 85,
50134 Florence, Italy
Received December 1, 2003
Polyfluorinated carbonic anhydrase inhibitors (CAIs) show very good inhibitory properties
against carbonic anhydrase (CA) and excellent in vivo antiglaucoma properties after topical
administration in rabbits. Still, the pentafluorinated compounds reported previously by this
group (Scozzafava et al. J . Med. Chem. 2000, 43, 4542-4551) showed high reactivity with thiol
groups of cysteine, glutathione, and presumably other proteins containing such moieties, which
may lead to severe ocular side effects. Here, we report an approach for obtaining fluorinated
CA inhibitors without the undesired enhanced reactivity. Thus, new compounds have been
obtained by attaching moieties with reduced reactivity toward aromatic substitution reactions
to the molecules of aromatic/heterocyclic sulfonamides possessing derivatizable amino moieties.
The employed tails of the 2,3,5,6-tetrafluorobenzoyl, 2,3,5,6-tetrafluophenylsulfonyl, and
pentafluorophenylureido types induced excellent CA inhibitory properties in the new reported
sulfonamides, mainly against the isozymes involved in aqueous humor secretion, CA II and
CA IV, whereas affinity for CA I was lower. Several low-nanomolar CA II inhibitors were
detected, which did not react with cysteine or glutathione, in contrast to the corresponding
perfluorinated compounds previously reported. These derivatives also showed a potent reduction
of the intraocular pressure (IOP) in hypertensive rabbits, amounting to 13-21 mmHg at 1 h
postadministration (compared to 5 mmHg obtained with dorzolamide, a clinically used drug),
and the decreased IOP was maintained for 4-5 h after the administration. These compounds
constitute valuable candidates for obtaining topically acting antiglaucoma CA inhibitors of a
new generation, with enhanced efficacy, prolonged duration of action, and reduced side effects.
In tr od u ction
advances in this field have then been achieved by the
Merck group, which discovered the first clinically used,
topically effective antiglaucoma sulfonamide, dorzola-
mide DZA.5 The approach for arriving at this compound
(and in fact also for brinzolamide BRZ, the second such
clinically used pharmacological agent)6 is known as the
“ring approach” because it involved the exploration of
a wide range of ring systems to which sulfamoyl
moieties were incorporated.2-4 We have explored an
alternative approach for the design of topically acting
antiglaucoma sulfonamides that consists of attaching
tails that will induce the desired physicochemical
properties (such as water solubility, enhanced penetra-
bility through the cornea, etc.) to scaffolds of aromatic/
heterocyclic sulfonamides also incorporating derivatiz-
able moieties of the amino/hydroxy type.2-4,7-10
Although the treatment of glaucoma with inhibitors
of the metalloenzyme carbonic anhydrase (CA, EC
4.2.1.1) is very effective in reducing elevated intraocular
pressure (IOP), the systemic administration of drugs
such as acetazolamide AAZ, methazolamide MZA, ethox-
zolamide EZA, and dichlorophenamide DCP leads to a
wide range of unpleasant side effects due to inhibition
of the enzyme present in tissues (kidneys, red cells,
stomach, etc.) other than the eye.1-4 The possibility of
the topical administration of the classical drugs from
this class mentioned above (acetazolamide, methazola-
mide, ethoxzolamide, or dichlorophenamide) was exten-
sively investigated by several researchers, but negative
results have been constantly obtained, and for more
than 40 years it was considered that CA inhibitors
(CAIs) could only be given systemically.1 Important
Many tails have been used for the design of topically
acting sulfonamide CAI antiglaucoma agents, such as
pyridinecarboxamido, quinolinesulfonylamido, polyami-
nopolycarboxamido, perfluoroalkyl/arylcarboxamido/
sulfonamido, aminoacyl, or oligopeptidyl among
others,2-4,7-10 but those leading to the most impressive
biological activity were the fluorine-containing ones.10
The constant search of novel topically acting antiglau-
* To whom correspondence should be addressed. Phone: +39-055-
4573005. Fax: +39-055-4573385. E-mail: claudiu.supuran@unifi.it.
† Natural and Synthetic Drugs Research Centre, Universita` degli
Studi di Firenze.
‡ University of Lie`ge.
§ University of Florence.
#
U.O. Oculistica Az. USL 3.
| Clinica Oculistica, Universita` degli Studi di Firenze.
10.1021/jm031116j CCC: $27.50 © 2004 American Chemical Society
Published on Web 04/20/2004

Carbonic Anhydrase Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 11 2797
those incorporating the perfluorinated aromatic moieties
(such as C6F5CO and C6F5SO2), a fact that was then
explained after the report of the high-resolution X-ray
crystal structure of isozyme CA II with one of these
agents, the perfluorobenzoylated analogue of meth-
azolamide, PFMZ (Figure 1).12
PFMZ is almost 10 times more effective a CA II
inhibitor (KI ) 1.5 nM) compared to methazolamide (KI
) 14 nM). Its binding to the enzyme active site was
shown to be similar to that of other sulfonamide
inhibitors,13,14 considering the interactions of the sul-
fonamide zinc anchoring group and thiadiazoline ring
contacts, but differs considerably when the perfluo-
robenzoylimino fragment of the molecule has been
analyzed. Thus, several unprecedented strong hydrogen
bonds involving the imino nitrogen, carbonyl oxygen, a
meta fluorine atom belonging to the inhibitor, and two
water molecules, as well as Gln 92 of the enzyme active
site, were evidenced. A stacking interaction of the
perfluorophenyl ring of the inhibitor and the aromatic
ring of Phe 131 was also observed for the first time in
a CA-sulfonamide adduct (Figure 1).12 All these find-
ings proved that the pentafluorophenyl tail of such CAIs
is indeed quite beneficial for obtaining very potent,
topically effective CAIs, but an undesired property of
such compounds was then discovered, related to the very
electrophilic character of the carbon atom bearing the
fluorine atom in the position para to the carboxamido/
sulfonamido moiety.15 Indeed, Medina et al.16,17 reported
a series of perfluorophenylsulfonamide derivatives of
types 16-21 (among which is T138067, compound 21
in phase II clinical trials for the treatment of human
cancers with MDR+ phenotype), which strongly inhibit
the growth of a variety of tumors, including multidrug-
coma sulfonamides is motivated by the rather wide
range of side effects associated with the two clinically
used drugs, dorzolamide and brinzolamide, which in-
clude local irritation and eye reddening, pruritus,
blurred vision, nephrolithiasis, anorexia, dementia,
corneal decompensation, etc.11
In a previous contribution from our laboratory,10 it
was shown that by attaching perfluoroalkyl/arylcar-
boxamido/sulfonamido tails, such as perfluorobutylsul-
fonyl, perfluorooctylcarboxamido, perfluorophenylcar-
boxamido, or perfluorophenylsulfonyl, to the aromatic/
heterocyclic sulfonamides also incorporating derivatizable
amino moieties, of types 1-15, very effective CAIs can
be obtained, which also showed good water solubility
and efficacy as topically acting antiglaucoma agents in
an animal model of the disease. The best CAIs and
antiglaucoma agents in that series of derivatives were
F igu r e 1. Schematic representation of PFMZ binding to the hCA II active site (distances in Å). Reprinted with permission from
ref 12. Copyright 2003 Taylor and Francis. see also http://www.tandf.co.uk.

2798 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 11
de Leval et al.
Sch em e 1
Ch a r t 1
resistant ones. Furthermore, the corresponding deriva-
tives possessing other halogens instead of the five
fluorine atoms of 16-21, together with the correspond-
ing isomeric trifluoro- and tetrafluorosulfonamides, were
much less active than the lead compound 16.16,17 This
was subsequently shown to be due to the mechanism of
action of these compounds (Scheme 1), which covalently
bind to Cys 239 of â-tubulin, by means of a nucleophilic
aromatic substitution reaction in which the most labile
fluorine atom of the antimitotic agent (i.e., the fluorine
in the position para to the sulfonamide moiety) reacts
with the thiol moiety of the Cys 239 residue of â-tubulin,
leading to modification of the protein.18
Resu lts
Syn th esis. The new derivatives A1-A8, A10-A14,
B3-B7, B10, B15, and C1-C10 described here were
prepared as previously described for related derivatives,
from the aminosulfonamides 1-15 and 2,3,5,6-tet-
rafluorobenzoyl chloride, 2,3,5,6-tetrafluorobenzene-
sulfonyl chloride, or pentafluorophenyl isocyanate, re-
spectively (Chart 1).10,23-25
Ca r bon ic An h yd r a se In h ibitor y Activity. The
new sulfonamides reported here were assayed for
inhibition of three CA isozymes, two of them known to
play a critical role in aqueous humor formation (CA II
and CA IV), whereas the other one, CA I, is known to
be important for the possible systemic side effects of
such drugs (Table 1).4
The enhanced lability of the p-fluorine group is
desired in antitumor agents of type 16-21 mentioned
above19,20 but is detrimental for topically acting CAIs
because of the fact that the eye fluids are very rich in
glutathione.21 In fact, many topically acting sulfonamide
CAIs could not be developed clinically just because of
the fact that their sulfonamido group was easily dis-
placed by reduced glutathione with formation of a
glutathione conjugate,21 which proved to be a potent
allergen, leading to eye irritations in experimental
animals.4,22 To avoid such problems, we disclose a new
class of potent, topically acting antiglaucoma CAIs
which, although possessing the favorable properties
associated with the perfluorophenyl-containing com-
pounds previously described,10 are devoid of enhanced
sensitivity to nucleophilic substitution and thus poten-
tial side effects due to reaction with glutathione or
cysteine-containing proteins.
Rea ction of F lu or in a ted Su lfon a m id es w ith Th i-
ol Rea gen ts. Reactivity of some of the newly synthe-
sized CAIs as well as other types of fluorinated sulfona-
mides with thiol reagents (cysteine and glutathione) is
shown in Table 2.
IOP Mea su r em en ts. In vivo IOP lowering data with
some of the most active CAIs reported here, in hyper-

Carbonic Anhydrase Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 11 2799
Ta ble 1. CA Inhibition Data with Standard Inhibitors and the
New Sulfonamides Reported in the Present Study
Sch em e 2
a
KI (nM)
inhibitor
hCA Ib
hCA IIb
bCA IVc
acetazolamide
methazolamide
ethoxzolamide
dichlorophenamide
dorzolamide
A1
A2
A3
A4
A5
A6
A7
A8
A10
A11
A12
A13
A14
B3
B4
B5
B6
B7
B10
B15
C1
C2
C3
C4
C5
C6
C7
C8
C9
C10
900 ( 42
780 ( 30
25 ( 1.8
12 ( 0.7
14 ( 1.5
8 ( 0.3
38 ( 3
220 ( 16
240 ( 13
13 ( 0.7
380 ( 24
43 ( 3
1200 ( 63
50000 ( 100
1500 ( 39
1700 ( 62
975 ( 18
980 ( 54
900 ( 68
760 ( 30
1080 ( 47
1870 ( 80
250 ( 13
270 ( 15
6300 ( 36
6100 ( 33
6000 ( 38
1300 ( 65
860 ( 47
750 ( 40
580 ( 39
930 ( 62
13 ( 1
320 ( 18
1750 ( 90
1800 ( 74
950 ( 53
1000 ( 76
940 ( 41
760 ( 33
1300 ( 76
1960 ( 150
740 ( 40
270 ( 18
9 ( 0.2
38 ( 3
690 ( 40
430 ( 13
400 ( 27
120 ( 5
94 ( 7
21 ( 1.5
16 ( 0.5
13 ( 0.6
12 ( 0.7
1.5 ( 0.1
76 ( 5
31 ( 2
42 ( 3
84 ( 6
50 ( 4
2.1 ( 0.2
1.4 ( 0.1
19 ( 2
24 ( 3
28 ( 2
63 ( 4
15 ( 1
54 ( 4
B10, and B15); (ii) replacement of the carboxamido/
sulfonamido linker between the perfluorinated aryl tail
and the aromatic/heterocyclic ring bearing the sulfamoyl
zinc anchoring group10 by a new linker that should
activate less the fluorine atom in the para position. We
opted for the ureido linker because of the ease of
preparing such compounds and because of the fact that
we have previously shown that these types of CAI show
high potency against the isozymes of interest for the
secretion of aqueous humor within the eye25 (Scheme
2). Furthermore, the ureido moiety is much less activat-
ing for aromatic nucleophilic substitution reactions
compared to the carboxamido or sulfonamido moieties.15
The carboxamides A1-A8 and A10-A14 were easily
prepared from the commercially available 2,3,5,6-tet-
rafluorobenzoic acid 22, which has been converted to
the corresponding acyl chloride 23 by treatment with
thionyl chloride, followed by reaction with the amino-
sulfonamides 1-8 and 10-14, in Schotten-Baumann
conditions, as previously reported.10,23 For the prepara-
tion of the sulfonamides B3-B7, B10, and B15, 1,2,4,5-
tetrafluorobenzene 24 was transformed to the mono-
sulfonyl chloride by treatment with chlorosulfonic acid
and thionyl chloride at 150 °C, followed by reaction with
the aminosulfonamides 3-7, 10, and 15, in Schotten-
Baumann conditions as previously reported.10,23,24 The
pentafluorophenyl ureas C1-C10 were easily obtained
from the commercially available isocyanate 26 and the
aminosulfonamides 1-10, as previously reported.25
In Vitr o CA In h ibition . CA inhibition data against
the three isozymes hCA I, hCA II, and bCA IV (h )
human, b ) bovine isozyme) for the prepared com-
pounds and standard inhibitors are shown in Table 1.
All the new compounds reported here act as better CAIs
than the parent sulfonamides from which they were
obtained (data not shown), showing a biological activity
quite similar to that of the corresponding perfluorinated
derivatives bearing the same carboxamido/sulfonamido
substitution pattern, as previously reported10 (data not
shown). Thus, the following SAR can be drawn from the
data of Table 1. (i) Considering the three tails A
(tetrafluorobenzoyl), B (tetrafluorophenylsulfonyl), and
C (pentafluorophenylureido), generally derivatives of
type B were more inhibitory than the corresponding
derivatives of type A, which in turn were more inhibi-
tory than the corresponding ureas of type C (but several
exceptions were detected such as the carboxamide A6,
13 ( 1
49 ( 5
25 ( 2
330 ( 16
135 ( 8
76 ( 6
12 ( 1
9 ( 0.4
3.8 ( 0.3
61 ( 5
42 ( 3
49 ( 2
0.7 ( 0.1
13 ( 1
44 ( 3
30 ( 2
15 ( 1
21 ( 1
485 ( 26
430 ( 35
21 ( 2
8 ( 0.4
15 ( 0.5
15 ( 1
50 ( 3
41 ( 4
18 ( 2
63 ( 5
80 ( 5
245 ( 13
360 ( 22
58 ( 4
95 ( 6
16 ( 1
3.6 ( 0.4
45 ( 2
a
Mean ( standard error (from three different assays). b Human
(cloned) isozymes c Bovine lung isozyme, by the esterase method.27
Ta ble 2. Reactivity against Thiol Reagents (Cysteine (Cys)
and Glutathione (Glt)) of Fluorinated Sulfonamide Derivativesa
% SH modificationb
compd
Cys
Glt
A6
B10
C3
27
28
16
0
0
0
55
76
98
0
0
0
49
68
96
a
The percent in the table means the amount of cysteine or
glutathione that reacted with the fluorinated sulfonamide inves-
tigated in the paper. After 24 h of incubation in a 1:1 molar ratio
of the reagents in phosphate buffer (see Experimental Protocols).
Errors were in the range 2-5% of the reported values, from three
different determinations.28
b
tensive rabbits (a widely used animal model of glau-
coma)26 after topical administration of the drug, are
shown in Figure 1.
Discu ssion
Ch em istr y. To prepare fluorine-containing CAIs
devoid of enhanced sensitivity to nucleophilic substitu-
tion, two approaches have been used: (i) design of
derivatives lacking the p-fluorine reactive group, with
two types of such derivatives being prepared (the
2,3,5,6-tetrafluorophenylcarboxamides A1-A8 and A10-
A14; the 2,3,5,6-tetrafluorophenylsulfonamides B3-B7,

2800 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 11
de Leval et al.
which is more inhibitory than the corresponding sul-
fonamide B6 against isozymes I and II, among others).
(ii) Considering the parent sulfonamides that were
derivatized by the three tails mentioned above, the
heterocyclic derivatives (such as the thiadiazoles and
thiadiazolines 10, 11) led to more potent inhibitors
compared to the benzenic derivatives. Among these last
benzenic derivatives, the best inhibitors were the sul-
fonylated sulfanilamide/homosulfanilamide/p-aminoet-
hylbenzenesulfonamide of types 12-14 (because of their
long molecules, a fact explained in QSAR24 and X-ray14b
crystallographic studies) and the fluorosulfanilamide
derivatives A6 and B6, whereas lower activity was
associated with the presence of the orthanilamide,
metanilamide, or chlorinated/brominated sulfanilamides
1, 2, 7, and 8 (the derivatives of these sulfonamides
appreciably inhibited these enzymes, with potencies
frequently comparable to that of dichlorophenamide, a
clinically used drug). (iii) Many of the new CAIs reported
here, such as A6, A10, A11, A14, B5, B6, B10, B15,
C3, and C10 among others, showed much better CA
inhibitory properties compared to the clinically used
drugs acetazolamide, methazolamide, dichlorophena-
mide, or dorzolamide. (iv) Isozyme hCA II was the most
susceptible to inhibition by the new compounds reported
here, generally followed by isozyme IV, whereas hCA I
was the less susceptible. This is a general behavior of
sulfonamides toward these three isozymes and is fre-
quently observed for other types of investigated sulfon-
amides2-4 (Table 1).
F igu r e 2. IOP lowering in hypertensive rabbits (initial
pressure in the range of 34 ( 1 mmHg) after topical treatment
with one drop (50 µL) of 2% solution of the CAIs dorzolamide,
A6, B10, and C3 (mean ( standard error, from three different
determinations).
As seen from the data of Figure 2, the three new CAIs
investigated in detail showed a potent and prolonged
reduction of IOP in hypertensive rabbits. Thus, dor-
zolamide (as hydrochloride salt), the standard drug,
induces a modest decrease of IOP after topical admin-
istration as a 2% water solution (pH 5.5), with the peak
at 1 h postadministration of around 5 mmHg. The effect
of this drug vanishes rapidly as the pressure returns to
basal levels after 2-2.5 h. The fluorinated sulfonamides
investigated here induced a very sharp decrease of IOP,
with reductions of 12-15 mmHg after 15 min postad-
ministration, with the maximal decrease of IOP gener-
ally also reaching the peak after 1 h postadministration
(the pH of these solutions were in the range 6.5-7.0,
being much less irritant than the dorzolamide hydro-
chloride solution). The maximal IOP reduction was
much larger compared to that of the standard drug
(dorzolamide), amounting to 21 mmHg for the best
compound (A6) and 13-15 mmHg for the other two
derivatives (B10 and C3) after 1 h. It is interesting to
note that the urea C3 showed its maximal activity not
at 1 h but at 2 h postadministration, when it reached a
decrease of IOP of 17.5 mmHg, maintaining reduced
IOP for another 2 h (data not shown). In fact, all these
CAIs showed a much more prolonged duration of action
compared to dorzolamide, of at least 4-5 h (data not
shown), similar to the pentafluorophenyl derivatives
previously reported.10 No signs of eye irritations have
been observed, even after prolonged administration of
the eye drops containing the new derivatives mentioned
above to the hypertensive rabbits used in these experi-
ments. Thus, this significant reduction of IOP, correlat-
ing with the good water solubility (in neutral form,
leading to nonirritating solutions with the pH in the
neutral range), and the lack of enhanced reactivity
toward thiol groups make these derivatives valuable
candidates for the development of the next generation
of topically acting antiglaucoma sulfonamides with
enhanced activity and reduced side effects.
Rea ction of F lu or in a ted Su lfon a m id es w ith Th i-
ol Rea gen ts. To validate the approach we designed for
obtaining CAIs devoid of undesired nucleophilicity, the
reactions of several new derivatives, the corresponding
pentafluorophenylsulfonamides previously reported 27
and 28,10 and the standard compound 16,16 with thiol
reagents (cysteine and glutathione) were investigated
(Table 2).28
As seen from the data of Table 2, the three new
compounds reported here and investigated for their
reactivity with thiol reagents, A6, B10, and C3, were
totally unreactive, whereas the corresponding perflu-
orinated CAIs 27 and 28 appreciably reacted with
cysteine as well as with glutathione after 24 h of
incubation. In the same conditions, the standard drug,
the antimitotic sulfonamide 16, completely reacted with
the two reagents. This clearly demonstrates that the
approach we propose here is a valid one for obtaining
fluorinated CAIs devoid of enhanced sensitivity toward
nucleophilic substitution.
IOP Mea su r em en ts. Some of the best in vitro CAIs
detected here, such as A6, B10, and C3, as well as
dorzolamide as standard drug, have been investigated
in vivo in hypertensive rabbits, a widely used animal
model of glaucoma, for their effects on intraocular
pressure (IOP) after topical administration as 2% water
solutions (Figure 2).10,26
Con clu sion s
Polyfluorinated CAIs have been obtained by attaching
fluorinated moieties with reduced sensitivity toward
nucleophilic substitution reactions to the molecules of

Carbonic Anhydrase Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 11 2801
was collected and stored at -20 °C. The purity of the final
compound was verified by TLC (MeOH/CHCl3, 3/7).
Gen er a l P r oced u r e for th e P r ep a r a tion of th e F lu or i-
n a t ed Su lfon a m id es. Met h od A (Sch ot t en -Ba u m a n n
Syn th esis). An amount of 5 mmol of sulfonamide 1-15 (such
as 5-amino-1,3,4-thiadiazole-2-sulfonamide 10 or 5-imino-4-
methyl-2-sulfonamido-δ2-1,3,4-thiadiazoline 11) was dissolved
in 15 mL of an aqueous 2.5 M solution of NaOH and cooled to
2-5 °C in a salt/ice bath. An amount of 5 mmol of sulfonyl/
acyl chloride 23 and 25 were added in small portions concomi-
tantly with 10 mL of a 2 M NaOH solution, maintaining the
temperature under 10 °C. The reaction mixture was then
stirred at room temperature for 5-10 h (TLC control), then
the pH was adjusted to 2 with 5 N HCl, and the precipitated
sulfonamides were filtered and recrystallized from aqueous
ethanol.
Meth od B. The aminosulfonamide 1-15 (1 g) was dissolved
in N,N-dimethylacetamide (10 mL), and 2 equiv of 2,3,5,6-
tetrafluorobenzenesulfonyl chloride 25 and 1 equiv of sodium
bicarbonate were added to the solution, which was left on ice
under stirring. After 60 min of stirring, water (50 mL) was
added to the solution, which was then extracted three times
with ethyl acetate (25 mL). The organic fractions were collected
and extracted three times with a 1 N hydrochloric acid aqueous
solution (15 mL). The organic phase was dried over magnesium
sulfate. After charcoal treatment, the organic phase was dried
under vacuum. The final compounds were recrystallized as
mentioned above. The purity of the final compound was
verified by TLC (MeOH/CHCl3, 3/7).
aromatic/heterocyclic sulfonamides possessing deriva-
tizable amino moieties. The employed tails of the 2,3,5,6-
tetrafluorobenzoyl, 2,3,5,6-tetrafluophenylsulfonyl, and
pentafluorophenylureido types induced excellent CA
inhibitory properties in the new sulfonamides reported
in the paper, mainly against the isozymes involved in
aqueous humor secretion, CA II, and CA IV, whereas
the affinity for CA I was lower. Several water-soluble,
low-nanomolar CA II inhibitors were detected, which
did not react with cysteine or glutathione, in contrast
to the corresponding perfluorinated compounds previ-
ously reported. These derivatives also showed a potent
reduction of IOP in hypertensive rabbits, amounting to
13-21 mmHg at 1 h postadministration (compared to
5 mmHg obtained with dorzolamide), and the decreased
IOP was maintained for 4-5 h without signs of eye
irritation. These compounds constitute valuable candi-
dates for obtaining topically acting antiglaucoma CAIs
of a new generation, with enhanced efficacy, prolonged
duration of action, and reduced side effects.
Exp er im en ta l P r otocols
Ch em istr y. Melting points were recorded with a heating
plate microscope and are not corrected. IR spectra were
recorded in KBr pellets with a Carl Zeiss IR-80 instrument.
1H NMR spectra were recorded in DMSO-d6 as solvent, with
a Bruker CPX200 or Varian 300 instrument. Chemical shifts
are reported as δ values relative to Me4Si as internal standard.
Elemental analyses were done by combustion for C, H, N with
an automated Carlo Erba analyzer and were (0.4% of the
theoretical values. All reactions were monitored by thin-layer
chromatography (TLC) using 0.25 mm precoated silica gel
plates (E. Merck). Analytical and preparative HPLC was
performed on a reversed-phase C18 Bondapack column with a
Hewlett-Packard instrument. Sulfonamides 1-15 used in the
synthesis were commercially available compounds (from Sigma-
Aldrich or Acros) or were prepared as described previously:
halogenosulfanilamides 6-8 by halogenation of sulfanilamide
as reported in the literature;29 compound 10 from acetazola-
mide by deacetylation;30 imine 11 by deprotection of meth-
azolamide with concentrated hydrochloric acid.31 The sulfona-
mide derivatives 11-14 were prepared as described in ref 32.
Perfluorosulfonyl halides, perfluoroacyl halides, 2,3,5,6-tet-
rafluorobenzoic acid, 1,2,4,5-tetrafluorobenzene, chlorosulfonic
acid, thionyl chloride, cysteine, glutatione, cystine, and oxi-
dized glutathione, as well as inorganic reagents were from
Sigma-Aldrich (Milan, Italy). Compound 16 was prepared as
described in ref 16 from 4-methoxyaniline and pentafluorophe-
nylsulfonyl chloride. Acetonitrile, N,N-dimethylacetamide (E.
Merck), or other solvents used in the synthesis were doubly
distilled and kept on molecular sieves in order to maintain
them in anhydrous conditions.
Meth od C. The sulfonamide to be derivatized was sus-
pended/dissolved in anhydrous acetonitrile (50 mL), and an
amount of 2 equiv of pentafluorophenyl isocyanate 26 was
added. The obtained mixture was stirred at room temperature
for 60 min. The solution was dried under vacuum, and the
obtained solid was recrystallized from aqueous ethanol. Purity
of the final compound was verified by TLC (MeOH/CHCl3, 3/7).
All compounds were extensively characterized. An example
from each subclass is shown below.
4-Meth yl-5-[2,3,5,6-tetr a flu or op h en ylca r boxim id o]-δ2-
1,3,4-th ia d ia zolin e-2-su lfon a m id e A11: white crystals, mp
202-3 °C; IR (KBr), cm-1, 1148 (SO2sym), 1373 (SO2as), 1554
(amide II N-H), 1613 (amide I CdO), 3060 (NH), 3365 (NH2
or N-H); 1H NMR (DMSO-d6), δ, ppm, 9.10 (br s, 1H, SO2-
NH), 8.13 (tt, H-4, 7.5, 10.3), 7.14 (br s, 2H, SO2NH2), 3.54 (s,
3H, Me); 13C NMR (DMSO-d6), δ, ppm, 31.5 (Me), 120.7, 124.3,
135.9, 164.91 (C-thiadiazoline), 164.94 (C-thiadiazoline); 19F
NMR (DMSO-d6), δ, ppm, -137.8 (2,6-F2), -158.2 (3,5-F2).
Anal. (C11H7F4N3O3S2) C, H, N.
4-[2,3,5,6-Te t r a flu or ob e n ze n e su lfon yla m id oe t h yl]-
ben zen esu lfon a m id e B5: colorless crystals, mp 187-8 °C;
IR (KBr) cm-1, 1154 and 1163 (SO2sym), 1350 and 1378 (SO2as),
3365 (NH, NH2).
Syn th esis of 2,3,5,6-Tetr a flu or oben zoyl Ch lor id e. An
amount of 2 equiv of tetrafluorobenzoic acid was treated with
5 equiv of thionyl chloride in 50 mL of anhydrous benzene.
The reaction mixture was heated at reflux for 5 h, then the
solvent and excess thionyl chloride were removed in vacuo,
and the acyl chloride was distilled under reduced pressure.
Syn th esis of 2,3,5,6-Tetr a flu or oben zen esu lfon yl Ch lo-
r id e. Chlorosulfonic acid (97 g, 55.36 mL) was added to 1,2,4,5-
tetrafluorobenzene (25 g), and the obtained solution was
heated at 150 °C. After 2 h, the heating was stopped in order
to allow the solution to reach room temperature and thionyl
chloride (39.96 g, 24.30 mL) was poured to the solution. The
resulting mixture was reheated for 3 h at 150 °C. The brown
solution obtained was then poured dropwise with stirring into
a mixture of 250 g of ice and 100 mL of water. The obtained
suspension was extracted three times with 50 mL of ethyl
acetate. The organic fractions were collected and dried over
anhydrous sodium sulfate and then concentrated by solvent
evaporation under vacuum. The obtained/resulting brown oil
1H NMR (DMSO-d6, δ ppm, J Hz), 8.79 (t, H-8, 5.5), 8.13 (tt,
H-4, 7.5, 10.3), 7.69 (d, H-13-15, 8.4), 7.38 (d, H-12-16, 8.4),
7.30 (s, NH2), 3.29 (bq, H-9, 5.5, 6.9), 2.83 (t, H-10, 6.9). H-4 is
coupled with the two F from positions 3 and 5, J ) 10.3 Hz,
as well as with the two F from positions 2 and 6, J ) 7.5 Hz.
N-H is coupled with CH2-9 (J ) 5.5 Hz), whereas CH2-9
appears as a broad quartet because it is also coupled with CH2-
10, which appears as a triplet (J (vic) ) 6.9 Hz). 13C NMR-
(DMSO-d6, δ ppm, J Hz), 120.87 (t, C-1, J (19F-13C) ) 15.3),
110.74 (t, C-4, J (19F-13C) ) 23.5), 145.63 (dddd, C-3, 4.4, 10.8,
15.2, 248.4), 142.85 (ddt, C-2, 3.3, 15.7, 254.5), 129.12 (C-12-
16), 125.49 (C-13-15), 142.59 (C-11 or C-14), 142.21 (C-14 or
C-11), 43.36 (C-9), 34.69 (C-10). C - 1 ) 128.5 + 15.5 - 2 ×
13.0 + 2 × 1.6 ) 121.2; C - 2 ) 128.5 + 34.8 - 13.0 + 1.6 -
4.4 ) 147.5; C - 3 ) 128.5 + 34.8 - 13.0 + 0.3 + 1.6 - 4.4 )
152.2; C - 4 ) 128.5 - 2 × 13.0 + 2 × 1.6 + 3.1 ) 108.8 ppm.

2802 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 11
de Leval et al.
(The detailed spectrum is presented because these compounds
show very complicated behavior because of the multiple
couplings between the protons and fluorine and carbon atoms
and no literature data for similar compounds are available as
far as we know). Anal. (C14H12F4N2O4S2) C, H, N.
5-P er flu or op h en ylu r eid o-1,3,4-t h ia d ia zol-2-su lfon a -
m id e C10: white crystals, mp 268-9 °C; IR (KBr), cm-1, 1151
and 1185 (SO2sym), 1360 and 1371 (SO2as), 3060 (NH), 3365
(NH2); 1H NMR (DMSO-d6), δ, ppm, 9.17 (br s, 2H, NHCONH),
7.25 (br s, 2H, SO2NH2); 13C NMR (DMSO-d6), δ, ppm, 124.9,
127.5, 138.6, 159.7 (C-2 of thiadiazole), 170.5 (C-5 of thiadia-
zole); 19F NMR (DMSO-d6), δ, ppm, -134.3 (2,6-F2), -143.9
(4-F), -158.6 (3,5- F2). Anal. (C9H4F5N5O3S23) C, H, N.
Ca r bon ic An h yd r a se In h ibition . Human CA I and CA
II cDNAs were expressed in Escherichia coli strain BL21 (DE3)
from the plasmids pACA/hCA I and pACA/hCA II described
by Lindskog’s group.33 Cell growth conditions were those
described in ref 34, and enzymes were purified by affinity
chromatography according to the method of Khalifah et al.35
Enzyme concentrations were determined spectrophotometri-
cally at 280 nm, utilizing a molar absorptivity of 49 mM-1 cm-1
for CA I and 54 mM-1 cm-1 for CA II based on Mr ) 28.85
kDa for CA I and 29.3 kDa for CA II.36,37 bCA IV was isolated
from bovine lung microsomes as described by Maren et al.,
and its concentration has been determined by titration with
ethoxzolamide.38
experiments (three animals were used for each inhibitor
studied). The experimental procedures conform to the Associa-
tion for Research in Vision and Ophthalmology Resolution on
the use of animals. The rabbits were kept in individual cages
with food and water provided ad libitum. The animals were
maintained on a 12 h/12 h light/dark cycle in a temperature-
controlled room at 22-26 °C. Solutions of inhibitors (2%, by
weight) were obtained in distilled-deionized water. The pH
of these solutions was in the range 5.5-8.4.
IOP was measured using a Tono-Pen XL tonometer (Medtron-
ic Solan) as described by Maren’s group.40,41 The pressure
readings were matched with two-point standard pressure
measurements at least twice each day using a Digilab calibra-
tion verifier. All IOP measurements were done by the same
investigator with the same tonometer. One drop of 0.2%
oxybuprocaine hydrochloride (novesine, Sandoz) diluted 1:1
with saline was instilled in each eye immediately before each
set of pressure measurements. IOP was measured three times
at each time interval, and the mean values were reported. IOP
was measured first immediately before drug administration
and then at 30 min after the instillation of the pharmacological
agent and then every 30 min for a period of 4-6 h. For all
IOP experiments, the drug was administered to only one eye,
leaving the contralateral eye as an untreated control. The
ocular hypotensive activity is expressed as the average differ-
ence in IOP between the treated and control eye, in this way
minimizing the diurnal, seasonal, and interindividual varia-
tions commonly observed in the rabbit.40,41 Ocular hypertension
was elicited in the right eye of albino rabbits by the instillation
of hydrocortisol (from Sigma) as described by Melena et al.26
The IOP of treated animals was checked daily, and after
approximately 3-4 weeks, an elevated pressure of 33-35
mmHg has been achieved. Such animals were used in the IOP
measurement experiments.
Initial rates of 4-nitrophenylacetate hydrolysis catalyzed by
different CA isozymes were monitored spectrophotometrically
at 400 nm with a Cary 3 instrument interfaced to an IBM
compatible PC.27 Solutions of substrate were prepared in
anhydrous acetonitrile; the substrate concentrations varied
between 2 × 10-2 and 1 × 10-6 M, working at 25°C. A molar
absorption coefficient ꢀ of 18 400 M-1 cm-1 was used for the
4-nitrophenolate formed by hydrolysis in the conditions of the
experiments (pH 7.40), as reported in the literature.27 Non-
enzymatic hydrolysis rates were always subtracted from the
observed rates. Triplicate experiments were done for each
inhibitor concentration, and the values reported throughout
the paper are the mean of such results. Stock solutions of
inhibitor (1 mM) were prepared in distilled-deionized water
with 10-20% (v/v) DMSO (which is not inhibitory at these
concentrations), and dilutions up to 0.01 nM were done
thereafter with distilled-deionized water. Inhibitor and en-
zyme solutions were preincubated together for 10 min at room
temperature prior to assay to allow for the formation of the
E-I complex. The inhibition constant KI was determined as
described by Pocker and Stone.27 Enzyme concentrations were
3.3 nM for CA II, 10 nM for CA I, and 34 nM for CA IV (this
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Mea su r em en t of Ton om etr ic IOP . Adult male New
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Carbonic Anhydrase Inhibitors
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